Citation: Tight Junction Protein Expression-Inducing Probiotics Alleviate TNBS-Induced Cognitive Impairment with Colitis in Mice

A leaky gut is closely connected with systemic inflammation and psychiatric disorder. The rectal injection of 2,4,6-trinitrobenzenesulfonic acid (TNBS) induces gut inflammation and cognitive function in mice. Therefore, we selected Bifidobacterium longum NK219, Lactococcus lactis NK209, and Lactobacillus rhamnosus NK210, which induced claudin-1 expression in TNBS- or lipopolysaccharide (LPS)-stimulated Caco-2 cells, from the fecal bacteria collection of humans and investigated their effects on cognitive function and systemic inflammatory immune response in TNBS-treated mice. The intrarectal injection of TNBS increased cognitive impairment-like behaviors in the novel object recognition and Y-maze tests, TNF-α, IL-1β, and IL-17 expression in the hippocampus and colon, and LPS level in the blood and feces, while the expression of hippocampal claudin-5 and colonic claudin-1 decreased. Oral administration of NK209, NK210, and NK219 singly or together decreased TNBS-impaired cognitive behaviors, TNF-α and IL-1β expression, NF-κB+Iba1+ cell and LPS+Iba1+ cell numbers in the hippocampus, and LPS level in the blood and feces, whereas BDNF+NeuN+ cell and claudin-5+ cell numbers and IL-10 expression increased. Furthermore, they suppressed TNBS-induced colon shortening and colonic TNF-α and IL-1β expression, while colonic IL-10 expression and mucin protein-2+ cell and claudin-1+ cell numbers expression increased. Of these, NK219 most strongly alleviated cognitive impairment and colitis. They additively alleviated cognitive impairment with colitis. Based on these findings, NK209, NK210, NK219, and their combinations may alleviate cognitive impairment with systemic inflammation by suppressing the absorption of gut bacterial products including LPS into the blood through the suppression of gut bacterial LPS production and alleviation of a leaky gut by increasing gut tight junction proteins and mucin-2 expression.


Introduction
A leaky gut, a breakdown of the gut physical barrier, increases the gut permeability with systemic inflammation [1,2]. The gut permeability is mainly dependent on the tight junction protein expression in the gastrointestinal tract, such as occludin, claudins, and zonula occludens, and mucus layer, such as mucin-2 [1,2]. The invasion of bacteria into the body causes a variety of inflammation such as colitis, chorioamnionitis, and cystitis, which suppresses the tight junction protein expression [3][4][5]. The suppression of tight junction protein and/or mucus layer expression by gut inflammation increases the absorption of gut bacterial antigens such as lipopolysaccharides (LPS) from the gastrointestinal tract into the blood, resulting in cognitive impairment with systemic inflammation [6][7][8].

Culture of Gut Bacteria
NK209, NK210, and NK219 were cultured in commercial media for probiotics including MRS broth (BD, Franklin Lakes, NJ, USA), centrifuged at 5000× g and at 4 • C for 20 min, and washed with saline [23,24]. The collected cells were used for experiments.

Culture of Caco-2 Cells
Caco-2 cells were cultured in DMEM containing 10% fetal bovine serum and 1% antibiotic-antimycotic at 37 • C, as previously reported [24]. The cells (1 × 10 5 cells/well) were incubated with probiotics (1 × 10 5 CFU/mL) in the absence or presence of TNBS or LPS in 12-well plate for 22 h and washed twice. The cells were collected and lysed in RIPA lysis buffer containing 1% phosphatase inhibitor cocktail and 1% protease inhibitor cocktail on ice. The claudin-1 expression level in the supernatant was measured, assessed by immunoblotting.

Animals
Male C57BL/6 mice (7-weeks old, 19-21 g) were purchased from Koatech Inc. (Seoul, Korea) and maintained under the controlled condition (temperature, 20-22 • C; humidity, 40-60% humidity; light/dark cycle, 12 h) and fed standard laboratory chow and water ad libitum. Mice were acclimatized for 1 week before experiments. All animal experiments were approved by the Kyung Hee University Institutional Animal Care and Use Committee of (IACUC No., KUASP(SE)-21451) and performed according to the Guide for University Laboratory Animals Care and Usage.
Cognitive behaviors were evaluated 2 h after the final probiotic treatment in the Y-maze task (YMT) and novel object recognition task (NORT). Mice were euthanized by cervical dislocation under the inhalation of CO 2 20 h after the behavioral tasks. Colon and brain tissues were collected for the analyses of ELISA and immunoblotting and stored at −80 • C.

Assessment of Cognitive Behavior Tasks
The YMT was performed in a three-arm horizontal maze (40 cm (length), 3 cm (width) and 12 cm (height)) for 8 min, as previously reported [19,23]. The spontaneous alteration (%) was described as a spontaneous to possible alternation ratio. The NORT was performed in a black rectangular open field maze (45 × 45 × 45 cm) for 10 min, as previously reported [17,19]. The exploration (%) was described as the new object-touching time to the old and neo-object-touching time ratio.

Immunofluorescence Assay
Mice were transcardially perfused, as previously reported [25,26]. Their brain and colon tissues were post-fixed, cytoprotected, frozen, sectioned, and washed [19,26]. The washed section was blocked with serum, incubated with primary antibodies for BDNF

Statistical Analysis
All data are described as means ± standard deviation (SD). The significance (p < 0.05) was analyzed by a one-way analysis of variance, followed by Kruskal-Wallis test with Dunn's post-hoc test, using GraphPad Prism 9 (GraphPad Software, Inc., San Diego, CA, USA).

Statistical Analysis
All data are described as means ± standard deviation (SD). The significance (p < 0.05) was analyzed by a one-way analysis of variance, followed by Kruskal-Wallis test with Dunn's post-hoc test, using GraphPad Prism 9 (GraphPad Software, Inc., San Diego, CA, USA).

NK209, NK210, and NK219 Suppressed TNBS-Induced LPS Levels in the Blood and Feces
The intrarectal injection of TNBS increased LPS level in the blood and feces, as previously reported [7] (Figure 4, Supplementary Figure S3). Treatment with NK209, NK210, or NK219 significantly decreased TNBS-induced LPS and TNF-α levels in the blood and LPS level in the feces.

NK209, NK210, and NK219 Suppressed TNBS-Induced LPS Levels in the Blood and Feces
The intrarectal injection of TNBS increased LPS level in the blood and feces, as previously reported [7] (Figure 4, Supplementary Figure S3). Treatment with NK209, NK210, or NK219 significantly decreased TNBS-induced LPS and TNF-α levels in the blood and LPS level in the feces.

Discussion
The gut bidirectionally communicates with the brain through the nervous, endocrine, and immune systems [27,28]. The induction of gut microbiota dysbiosis by stressors such as pathogens and antibiotics triggers an inflammatory response, suppresses tight junction protein expression, and exacerbates systemic inflammation including neuroinflammation and colitis through an increased gut permeability, which easily translocate gut bacteria and their byproducts such as LPS into the body [9,25,[29][30][31]. Bacterial LPS is a strong mediator of inflammation [9,[32][33][34]. The translocation of LPS into the blood stimulates brainblood barrier endothelial, leptomeningeal, and microglial cells, leading to psychiatric diseases with neuroinflammation [9,30,34].

Discussion
The gut bidirectionally communicates with the brain through the nervous, endocrine, and immune systems [27,28]. The induction of gut microbiota dysbiosis by stressors such as pathogens and antibiotics triggers an inflammatory response, suppresses tight junction protein expression, and exacerbates systemic inflammation including neuroinflammation and colitis through an increased gut permeability, which easily translocate gut bacteria and their byproducts such as LPS into the body [9,25,[29][30][31]. Bacterial LPS is a strong mediator of inflammation [9,[32][33][34]. The translocation of LPS into the blood stimulates brain-blood barrier endothelial, leptomeningeal, and microglial cells, leading to psychiatric diseases with neuroinflammation [9,30,34].
We found that the intrarectal injection of TNBS in mice caused colonic inflammation and decreased the expression of claudin-1, a tight junction protein, and mucin-2, a member of mucin proteins, in the colon. Moreover, TNBS treatment increased LPS level in the blood and feces. These results suggest that TNBS treatment can induce LPS-absorbable colitis (a leaky gut). Treatment with TNBS increased cognitive impairment-like behaviors with neuroinflammation, as previously reported [9]. Moreover, TNBS increased hippocampal NF-κB + Iba1 + cell and LPS + Iba1+ cell numbers, while hippocampal BDNF + NeuN + cell and claudin-5+ cell numbers decreased. Jang et al. reported that TNBS treatment impaired cognitive function and suppressed BDNF expression in the hippocampus [9]. Stressors including diets with high fat, antibiotics, immobilization stress, and TNBS increase gut bacterial LPS in the intestine [9,25,[35][36][37]. Gut bacterial LPS induces gut inflammation, which increases gut permeability of microbial products such as LPS [25,37]. We also found that TNBS treatment increased fecal and blood LPS levels in mice and decreased the expression of tight junction protein claudin-1 in the colon and claudin-5 in the hippocampus. Exposure to LPS causes neuroinflammation and neurodegeneration in vivo [25,34]. These results suggest that TNBS treatment may cause cognitive impairment with neuroinflammation by inducing hippocampal NF-κB activation and suppressing LPS-hippocampal BDNF expression through an increase in the brain-blood barrier permeability of LPS.
However, NK209, NK210, and NK219 singly or together suppressed TNBS-induced expression of TNF-α and IL-1β in the colon and hippocampus, while IL-10 expression increased. They increased TNBS-suppressed claudin-1 and mucin barrier protein mucin-2 expression in the colon and claudin-5 in the hippocampus, resulting in the attenuation of TNBS-induced colitis and neuroinflammation. These probiotics also decreased blood and fecal LPS levels. They also increased claudin-1 expression in TNBS-stimulated Caco-2 cells in vitro. These findings suggest that NK209, NK210, and NK219 may singly or together mitigate colitis and neuroinflammation by increasing mucin-2 and tight junction protein expression and decreasing fecal LPS production.

Conclusions
TNBS induced gut inflammation with increased LPS-permeable leaky gut and impaired cognitive function. NK209, NK210, NK219, and their combinations may alleviate cognitive impairment with systemic inflammation by suppressing the absorption of gut bacterial products including LPS into the blood through the suppression of gut bacterial LPS production and alleviation of a leaky gut by increasing gut tight junction proteins and mucin-2 expression.

Conflicts of Interest:
The authors declare no conflict of interest.